doi:10.1016/j.chroma.2006.11.003 
Copyright © 2006 Elsevier B.V. All rights reserved.
Hydrophobic interaction chromatography of proteins IV Kinetics of protein spreading
Emmerich Haimera, Anne Tscheliessniga, Rainer Hahna and Alois Jungbauer
, a, 
aDepartment of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Received 1 May 2006;
revised 24 October 2006;
accepted 1 November 2006.
Available online 20 November 2006.
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Abstract
Adsorption of proteins on surfaces of hydrophobic interaction chromatography media is at least a two-stage process. Application of pure protein pulses (bovine serum albumin and β-lactoglobulin) to hydrophobic interaction chromatography media yielded two chromatographic peaks at low salt concentrations. At these salt concentrations, the adsorption process is affected by a second reaction, which can be interpreted as protein spreading or partial unfolding of the protein. The kinetic constants of the spreading reaction were derived from pulse response experiments at different residence times and varying concentrations by applying a modified adsorption model considering conformational changes. The obtained parameters were used to calculate uptake and breakthrough curves for spreading proteins. Although these parameters were determined at low saturation of the column, predictions of overloaded situations could match the experimental runs satisfactorily. Our findings suggest that proteins which are sensitive to conformational changes should be loaded at high salt concentrations in order to accelerate the adsorption reaction and to obtain steeper breakthrough curves.
Keywords: Hydrophobic interaction chromatography; Adsorption; Protein spreading
Fig. 1. Schematical description of adsorption and spreading of proteins. For irreversible spreading, the system reduces to three reaction constants (k1, k−1, k2).
Fig. 2. Pulse response experiments of BSA (A) and β-lactoglobulin (C) on Toyopearl Butyl 650 M at varying flow rates and evaluation of the isocratic BSA (B) and β-lactoglobulin (D) pulses. Plots of q2/q1 versus qxt with slope k2.
Fig. 3. Pulse response experiments for BSA (A) and β-lactoglobulin (C) at varying protein concentrations. BSA (B) and β-lactoglobulin (D) spreading in dependency of protein concentrations. For β > 1 increasing pulse concentrations lead to decreasing protein spreading due to sterical limitations.
Fig. 4. Comparison of calculated and measured uptake curve for BSA (A) and β-lactoglobulin (B) at 0.7 m (NH4)2SO4. Uptake was measured by the dip probe method. Grey dots indicate dip probe data [22].
Fig. 5. Calculated (°) and measured (-) BTC for β-lactoglobulin (A) and BSA (B) at 0.7 m (NH4)2SO4.
Fig. 6. (A) Effects of the spreading rate constant and the beta value on the elution front shape and (B) on the uptake curve.
Fig. 7. (A) BTC for spreading (BSA, β-lactoglobulin) and non-spreading (lysozyme) proteins in the region of same order or magnitude for adsorption and spreading reaction – the critical salt concentration and (B) below the critical salt concentration.
Fig. 8. Uptake curves for BSA (A) and β-lactoglobulin (B) at 0.7 m (NH4)2SO4 and 1.2 m (NH4)2SO4 (grey dots indicate dip probe data/lines are smoothed data).
Fig. 9. Experimental and uptake curves for BSA (A) and β-lactoglobulin (C). Total native and spreaded protein has been calculated and superimposed. In (B), the k1 value has arbitrarily increased by a factor of 100 and in (D) β was reduced from 3.8 to 1.
Fig. A.1. Quadratic base functions defined in the local (x) and global (z) coordinates.
Table 1.
Model parameters for Eqs. (3), (4) and (5).
Parameter k2 was calculated from isocratic pulse response data performed at different residence times and applying Eq. (9). Parameters k1, k−1 were calculated from isocratic pulse response data performed at different protein concentrations and applying Eq. (9). BSA and β-lactoglobulin. q was determined from batch uptake experiments.
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